Ribonucleic acid (RNA) purified from biological material is utilized extensively for molecular biology research and is becoming an important tool in human clinical testing. Most commonly, the isolated RNA is characterized by size and quantity to provide diagnostic information about both normal and aberrant functioning of genes. For example, gross DNA rearrangements associated with common leukemias are detected by isolation and identification of abnormal, hybrid RNAs.
Typically, there are three aspects of isolating substantially undegraded RNA from biological samples: (1) the cells or viral protein coats are lysed to release RNA; (2) ribonucleases (RNases) are inactivated to prevent RNA degradation; and (3) contaminants are removed to purify the preparation. Because of the abundance and stability of RNases in biological materials, it is important that cell or protein coat lysis and RNase inactivation be substantially simultaneous. Therefore, in its simplest form, the isolation of RNA is reduced to just two main steps: (1) cell lysis (or protein denaturation)/RNase inactivation; and (2) RNA purification.
Several lysing reagents have been formulated to lyse cells and/or viral protein coats and inactivate RNases substantially simultaneously. A lysate is created by mixing suspended cells (or biological fluid) with the lysing reagent, or by grinding tissues with a pestle in the presence of the lysing reagent, which facilitates penetration of the lysing reagent. The lysate reagent typically contains a detergent to dissolve cells and to solubilize proteins and lipids. A strong protein denaturant (i.e., denaturing agent) is usually added to aid in inactivating RNases. In addition, a strong reductant is often included to ensure complete protein denaturation.
The most common detergents used in lysing reagent formulations are the anionic detergents sodium dodecyl sulfate (SDS) and N-lauroyl sarcosine as described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., 7.3-7.24, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) and Ausubel, et al., Current Protocols in Molecular Biology., 4.0.4-4.5.3 and 13.12.1-13.12.3, John Wiley & Sons, New York (1989). Also, nonionic and cationic detergents have been described for this purpose by Favaloro et al., Methods Enzymol., 65, 718-749 (1980) and Macfarlane, (U.S. Pat. No. 5,010,183), respectively. Typically, nonionic detergents are undesirable because they are generally ineffective at inactivating RNases in tissues with high nuclease activity. Cationic detergents are generally undesirable because they are more hazardous than nonionic and anionic detergents. For example, the rat intravenous LD50 is 1200 mg/kg for the nonionic detergent Triton X-100 and 118 mg/kg for the anionic detergent SDS, but only 6.8 mg/kg for the cationic detergent dodecyltrimethylammonium bromide.
Strong protein denaturants are commonly added to the lysing reagent to ensure inactivation of RNases. The most effective and widely used is guanidinium thiocyanate, which is described by Ullrich et al., Science, 196, 1313-1319 (1977) and Chirgwin et al., Biochemistry, 19, 5294-5299 (1979). Less commonly used as RNase inhibitors are organoclays, which are described by Ness et al. (U.S. Pat. No. 5,393,672).
Other denaturing agents that have been used are guanidine hydrochloride and urea, which are described by Cox in Methods Enzymol., 12, 120-129(1968) and Auffray et al., Eur. J. Biochem., 107, 303-314(1980), respectively. These denaturing agents, however, are less effective at inactivating RNases than guanidinium thiocyanate. The addition of a proteolytic enzyme, such as Proteinase K, to digest RNases is another strategy used in RNA isolation techniques. This also is less effective than guanidinium thiocyanate because it is generally too slow at inactivating RNases causing RNA degradation, particularly in solid tissue preparations.
In addition to this primary denaturant, it is common practice to add a second denaturant, such as the sulfhydryl reducing agent 2-mercaptoethanol, to the lysing reagent to ensure complete protein denaturation. This denaturant is highly toxic and has a pungent odor, and is therefore not easy to use. Furthermore, it is also subject to oxidative degradation and therefore reduces the shelf-life of lysing reagents.
An important factor to consider in the formulation of lysing reagents is pH. It has been shown by Noonberg et al., BioTechniques, 19, 731-733 (1995) that for lysing reagents containing organic solvents, the lower the pH, the lower the degree of RNA degradation, within the pH range of 5.5 to 8.0. However, a review of RNA isolation methods indicates that the pH of the lysing reagent is no lower than 4.0 (Chomczynski, U.S. Pat. No. 4,843,155), and can be as high as 9.0 (Bugos et al., BioTechniques 19, 734-737 (1995), with most in the neutral range of 7.0-8.0. Chomczynski teaches, however, that a pH of lower than 4 results in a significantly lower degree of RNA isolation.
After cell or protein coat lysis and RNase inactivation, RNA is purified by isolating it from the complex lysate. There are two general strategies in widespread use for liquid phase purification of RNA. These are differential centrifugation and solvent extraction combined with salt precipitation.
To separate RNA from deoxyribonucleic acid (DNA) and protein contaminants using differential centrifugation, typically the lysate is placed onto a solution of cesium chloride as described by Glisin et al., Biochem., 13, 2633-2637 (1974) and Chirgwin et al., Biochemistry, 19, 5294-5299 (1979). Then the sample is centrifuged at high speed (at least 130,000.times. g) for at least 12 hours to selectively sediment the RNA, leaving contaminants in the supernatant fraction. This method has the disadvantages of being very time-consuming, requiring the use of expensive ultracentrifugation equipment, and it does not efficiently recover low molecular weight RNAs, such as 5S ribosomal RNAs and transfer RNAs.
The second strategy for RNA purification is to mix the lysate with both an organic solvent (typically, phenol and chloroform) and a salt (typically, sodium acetate). Phenol not only denatures proteins but, following centrifugation, causes the protein to collect at the interface between the organic and aqueous layers. Chloroform facilitates the separation of organic and aqueous phases. Such phenol-based reagents, however, are typically unstable during storage due to oxidation.
At low pH (e.g., 4-7), the addition of a high concentration (e.g., 2-3 molar) salt solution causes DNA to selectively precipitate so that following centrifugation, it too will collect at the organic-aqueous interface. Thus, by combining the phenol extraction with salt precipitation, both proteins and DNA collect at the interface following centrifugation, leaving RNA in the supernatant. This is described, for example, by Chomczynski et al., Anal. Biochem., 162 156-159 (1987) and Chomczynski, EP 0 554 034.
The salt solutions generally used in solvent extraction-salt precipitation techniques are typically sodium acetate solutions of pH 4.0 to pH 7.0 at concentrations of 2-3 molar. An alternative salt, lithium chloride, selectively precipitate RNA rather than the contaminating DNA. The addition of this salt to the aqueous fraction, recovered after phenol-chloroform extraction, is described by Ausubel et al., Current Protocols in Molecular Biology, 4.0.4-4.5.3. John Wiley & Sons, New York (1989) and Auffray et al., Eur. J. Biochem., 107, 303-314 (1980). However, a disadvantage of lithium chloride precipitation is that the low molecular weight RNAs are not recovered.
Reagents required for isolating RNA in conventional methods are formulated typically using organic solvents and other generally hazardous chemicals. For example, the raw materials in wide use are listed below, along with label precautions and toxicity information as obtained from Sigma Chemical Company. The toxicity data are given as LD50 values where the lower the LD50 value, the more hazardous the compound. Generally, lysing and/or purification solutions contain: chloroform, which is highly toxic and may cause cancer, having an LD50 of 908 mg/kg (rat oral administration); guanidinium thiocyanate, which is considered harmful, having an LD50 of 300 mg/kg (mouse intraperitoneal injection); 2-mercaptoethanol, which is considered highly toxic and has a very strong odor stench, having an LD50 of 244 mg/kg (rat oral administration); and phenol, which is highly toxic, having an LD50 of 317 mg/kg (mouse oral administration).
A method for DNA and RNA isolation that uses less hazardous compounds, such as benzyl alcohol to replace phenol and chloroform, is disclosed by Ness et al., U.S. Pat. No.5,393,672. Despite the lower toxicity of benzyl alcohol, it is still classified as harmful with an LD50 of 1230 mg/kg by rat oral administration. In addition, even less toxic organic solvents require special handling and disposal.
Thus, there is a need in the field for a method that is less hazardous and/or does not involves the use of organic solvents. In addition, there is a need for reagents that are more stable at room temperature (i.e., 20-30.degree. C.). Also, there is a need for relatively rapid protocols to isolate RNA from a variety of biological materials, especially for routine testing as found in clinical laboratories.